We use Drosophila as a model for addiction studies by studying tolerance, a core endophenotype of addiction. Here tolerance is defined as a reduced response to a drug caused by previous drug exposure. Drosophila acquire tolerance to the sedative effects of ethanol and organic solvent anesthetics such as benzyl alcohol after a single exposure. Previous work in our lab showed that induction of the slo gene, which encodes a BK channel, underlies functional tolerance: slo mutants fail to acquire tolerance, and induction of a slo transgene phenocopies tolerance. Analysis of the slo control region identified three conserved CRE sites, implicating the CREB family of transcription factors in slo induction and the response to drug sedation. In invertebrates and mammals CREB has been shown to be involved in learning and memory, depression, anxiety, circadian rhythms and endophenotypes of addiction, with evidence for roles in drug reward, tolerance and withdrawal. This evidence led our group to investigate the role of the two Drosophila CREB genes, CrebA and Creb2, in the acquisition of tolerance. We found that after sedation CrebA expression increases, a CREB repressor isoform (Creb2b) is downregulated, CREB occupancy at the slo control region increases, and a CRE-regulated reporter gene is induced. Additionally, Creb2 mutants fail to induce slo or acquire tolerance, and transgenic induction of the Creb2b repressor isoform blocks tolerance and slo induction (Wang et al. 2007, 2009). It is clear that CREB signaling is important for the acquisition of tolerance in Drosophila, but the role of CrebA has not been described. To explore the role of CrebA in the acquisition of tolerance, we will test mutant, RNAi expressing and transgenic flies for their ability to acquire tolerance to ethanol. Using real-time RT-PCR and chromatin immunoprecipitation with an epitope-tagged CrebA gene, we will determine if the role of CrebA in the acquisition of tolerance is to regulate of slowpoke following sedation. PUBLIC HEALTH RELEVANCE: The goal of this research is to describe genes involved the neuronal adaptations that lead to alcohol tolerance. Understanding of the mechanisms responsible for the acquisition of tolerance will lead to better understanding of the neuronal changes that lead to dependence and alcoholism.